Original Article
Treatment of brain glioblastoma multiforme with pcDNA3.1‑Egr. 1p‑p16 combined with gamma knife radiation: An experimental study on nude mice Liu Wenke, Li Peng*, Wang Xing, Shi Yujun1, Zhong Qi, Ren Haibo, Wang Wei Department of Neurosurgery, West China Hospital, Sichuan University, , 1The Key Laboratory of Transplant Engineering and Immunologyof the Ministry of Health, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China
Abstract
Address for correspondence: Dr. Wei Wang, Department of Neurosurgery, West China Hospital, Sichuan University, No. 37, Guo Xue Xiang, Chengdu - 610041, Sichuan, China. E‑mail: [email protected] Received : 03‑07‑2012 Review completed : 26‑08‑2012 Accepted : 20‑10‑2013
Background: High post‑operative recurrence and poor prognosis are likely to be related to the infiltrative growth of the glioblastoma multiforme (GBM). Objectives: The primary objective of this study is to investigate the possible synergistic effect of the combined treatment of gamma knife radio‑surgery (GKRS) and gene therapy for GBM and secondary objective is to explore the role of GKRS for the temporal and spatial regulation of the gene expression. Materials and Methods: The study performed on 70 nude mice and randomly divided into seven groups. Subcutaneous injection of human GBM tumor cells (T98G) was carried out to establish the animal models. Various doses of liposome‑mediated pcDNA3.1‑Egr. 1p‑p16 recombinant plasmid were transfected through intra‑tumor injection. GKRS was scheduled following the plasmid transfection. Tumor volumes were measured every 4 days after the treatment. Subcutaneous tumor nodule specimens were collected to analyze the cell apoptosis and p16 gene expression using terminal‑deoxynucleoitidyl transferase mediated nick end labeling staining and reverse transcription‑polymerase chain reaction. Tumor volumes, levels of cell apoptosis and p16 gene expression were compared between groups. Results: Rates of tumor growth were significantly lower in the pcDNA3.1‑Egr. 1p‑p16 plasmid + GKRS groups than that in the remaining groups 28 days following the GKRS management. The p16mRNA expression was noted in both of the pcDNA3.1‑Egr. 1p‑p16 plasmid group and the pcDNA3.1‑Egr. 1p‑p16 plasmid + GKRS with marginal‑dose of 20 Gy group. The level of messenger ribonucleic acid expression was higher in the pcDNA3.1‑Egr. 1p‑p16 plasmid + GKRS with the marginal‑dose of 20 Gy group, with a markedly increased apoptotic and necrotic cells, than that in the pcDNA3.1‑Egr. 1p‑p16 plasmid group. Conclusions: In animal studies, pcDNA3.1‑Egr. 1p‑p16 in combination with GKRS is a preferable management option for the GBM to the sole use of GKRS or gene therapy. It may be a novel approach for the treatment of human patient with GBM.
Access this article online Quick Response Code:
Website: www.neurologyindia.com PMID: *** DOI: 10.4103/0028-3886.121917
Neurology India | Sep-Oct 2013 | Vol 61 | Issue 5
Key words: Gamma knife, gene therapy, glioblastoma
multiforme, recombinant plasmid pcDNA3.1‑Egr. 1p‑p16
Introduction The annual incidence rate of glioblastoma is about 5/100,000 persons, 60‑70% of which are glioblastoma multiforme (GBM). [1] Integrated treatment of 491
Wenke, et al.: Treatment of glioblastoma multiforme with gamma knife and PcDNA3.1‑Egr. 1p‑p16
surgical resection combined with radiotherapy and chemotherapy is the mainstream therapeutic solution in GBM. However, high postoperative recurrence and poor prognosis, which is likely due to the infiltrative growth of the tumor and it also effects the quality of life the patients. Combined usage of tumor suppressor gene p16 with radiation‑induced promoter Egr‑1p is new approach of gene and radiation therapy for GBM. In our experimental study, pcDNA3.1‑Egr. 1p‑p16 plasmid is innovatively combined with gamma knife radio‑surgery (GKRS) for the treatment of brain GBM, which has not been reported before. Our study is to investigate the possible synergistic effect of the combined treatment of GKRS and gene therapy for GBM and to explore the role of GKRS for the temporal and spatial regulation of the gene expression.
Materials and Methods Reagents and apparatus 1. pcDNA3.1‑Egr. 1p‑p16 plasmid was constructed by GenScript Co. Ltd.; 2. Human GBM cell line T98G was provided by State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University; 3. 4‑6 weeks aged BALB/c‑nu/nu female nude mice with the weight of approximately 20 g were purchased from Chengdu Dashuo Biosciences Co. Ltd.; 4. Plasmid Midi Kit was from Qiagen Co. Ltd.; 5. In situ terminal‑deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) array kit was from Roche Co. Ltd.; 6. Lipofectamine 2000 was from Invitrogen Co. Ltd.; 7. Magnetic resonance imaging scanner, SIEMENS Sonata 1.5T; 8. Gamma Knife, Leksell Gamma Knife Model C, Elekta Co. Ltd. Experiment methods Cell thawing and cultivation Frozen cells were removed from liquid nitrogen and quickly placed in 37°C water bath for rewarming and thawing. The cells were washed once with Dulbecco’s modified eagle medium (DMEM) and then centrifuged at 1500 rpm for 5 min. The supernatant was removed and the cells were then cultivated on the complete DMEM at 37°C in a 5% CO2 incubator. Cell passage The medium was removed and the cells were washed twice with pre‑warmed serum‑free DMEM and digested by 1% trypsin‑ethylene diamine tetraacetic acid. The digestion was terminated by adding complete DMEM when the cells became smaller and round under the microscope. After the cells were blown 492
scattered, the liquid was collected and centrifuged at 1500 rpm for 5 min. The cells were re‑suspended in the complete medium after precipitation and divided into flasks for continuous cultivation. When grew to a certain quantity, the well grown cells were collected and washed with sterile phosphate buffer solution (PBS). The cells were counted on a counting chamber under microscope to adjust the concentration to 5 × 107 cells/ml. Preparation of plasmid The bacterial strain for converting the pcDNA3.1‑Egr. 1p‑p16 recombinant plasmid was inoculated in the Luria‑Bertani medium and then cultivated at 37°C on a shaker overnight. The plasmid was extracted by the alkaline lysis method with modification. Restriction enzyme digestion was performed on collected samples for confirmation. Animal model establishment A total of 70, 4‑6 weeks aged BALB/c‑nu/nu nude mice, weighing approximately 20 g each, were used for the experimental study. Each nude mouse was inoculated with 5 × 106 T98G cells (0.1 ml) subcutaneously under the right axilla. An equal volume of PBS was subcutaneously injected under the right axilla of the control nude mice. The animals were treated i when the tumors in the mice of the experimental group grew up to 5 mm in diameter. The grouping and treatments are detailed in Table 1. Post‑modeling treatments The liposome‑encapsulated pcDNA3.1‑Egr. 1p‑p16 recombinant plasmid was transfected to the tumor cells by intra‑tumor injection. The nude mice were anesthetized by intra‑peritoneal injection of 3% pentobarbital sodium (40 mg/kg) 48 h after transfection. The animal was fixed on a previously prepared polymethyl methacrylate made fixation device. Then the device was fixed onto the Leksell stereotactic head frame [Figure 1]. The radiation procedure was finished using Leksell Gamma Knife Model C under the guidance of magnetic resonance imaging (Siemens sonata 1.5T). According to the grouping rules, the tumor was covered with the established isodose line [Figure 2]. The radiation doses are detailed in Table 1. Observation of anti‑tumor effect Tumor volume was recorded by measuring its length (a) and width (b) using a vernier caliper every 4 days after the GKRS. Tumor growth rate and volume were calculated based on the formulas: tumor growth rate f = post‑radiation tumor volume/pre‑radiation tumor volume; tumor volume V (mm3) = (a × b2)/2. The data were presented in x ± s. To compare the tumor volume changes, a t‑test was used. Statistical significance was considered at a P ≤ 0.05. Neurology India | Sep-Oct 2013 | Vol 61 | Issue 5
Wenke, et al.: Treatment of glioblastoma multiforme with gamma knife and PcDNA3.1‑Egr. 1p‑p16
Figure 2: Gamma knife treatment protocol: the tumor foci was covered by the isodose line
Figure 1: Gamma knife treatment status
Table 1: Grouping of experiment animal and tumor growth rate
Group
Group code
No. of animal
Radiation Null plasmid group+radiation Plasmid group 1
R Palone+R
10 10
P1
10
Plasmid group 2
P2
10
Plasmid group 1+20 Gy radiation Plasmid group 2+20 Gy radiation Control group
P1+R
10
P2+R
10
Control
10
Treatment
Tumor growth rate
Sole 20 Gy marginal‑dose radiation Intra‑tumoral injection of 20 µg pcDNA 3.1+plasmid encapsulated in 50 µl liposome+20 Gy marginal‑dose radiation Intra‑tumoral injection of 20 µg pcDNA3.1‑p16 plasmid encapsulated in 50 µl liposome Intra‑tumoral injection of 20 µg pcDNA3.1‑Egr. 1p‑p16 plasmid encapsulated in 50 µl liposome Intra‑tumoral injection of 20 µg pcDNA3.1‑p16 plasmid encapsulated in 50 µl liposome+20 Gy marginal‑dose radiation Intra‑tumoral injection of 20 µg pcDNA3.1‑Egr. 1p‑p16 plasmid encapsulated in 50 µl liposome+20 Gy marginal‑dose radiation Intra‑tumoral injection of 50 µl PBS, without radiation
3.51±0.88 3.40±0.7 3.83±1.11 3.85±0.65 1.95±0.35 1.00±0.21 5.44±1.02
Each group was individually compared with the control group and the differences in tumor growth rate were statistically significant (P
Treatment of brain glioblastoma multiforme with pcDNA3.1‑Egr. 1p‑p16 combined with gamma knife radiation: An experimental study on nude mice Liu Wenke, Li Peng*, Wang Xing, Shi Yujun1, Zhong Qi, Ren Haibo, Wang Wei Department of Neurosurgery, West China Hospital, Sichuan University, , 1The Key Laboratory of Transplant Engineering and Immunologyof the Ministry of Health, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China
Abstract
Address for correspondence: Dr. Wei Wang, Department of Neurosurgery, West China Hospital, Sichuan University, No. 37, Guo Xue Xiang, Chengdu - 610041, Sichuan, China. E‑mail: [email protected] Received : 03‑07‑2012 Review completed : 26‑08‑2012 Accepted : 20‑10‑2013
Background: High post‑operative recurrence and poor prognosis are likely to be related to the infiltrative growth of the glioblastoma multiforme (GBM). Objectives: The primary objective of this study is to investigate the possible synergistic effect of the combined treatment of gamma knife radio‑surgery (GKRS) and gene therapy for GBM and secondary objective is to explore the role of GKRS for the temporal and spatial regulation of the gene expression. Materials and Methods: The study performed on 70 nude mice and randomly divided into seven groups. Subcutaneous injection of human GBM tumor cells (T98G) was carried out to establish the animal models. Various doses of liposome‑mediated pcDNA3.1‑Egr. 1p‑p16 recombinant plasmid were transfected through intra‑tumor injection. GKRS was scheduled following the plasmid transfection. Tumor volumes were measured every 4 days after the treatment. Subcutaneous tumor nodule specimens were collected to analyze the cell apoptosis and p16 gene expression using terminal‑deoxynucleoitidyl transferase mediated nick end labeling staining and reverse transcription‑polymerase chain reaction. Tumor volumes, levels of cell apoptosis and p16 gene expression were compared between groups. Results: Rates of tumor growth were significantly lower in the pcDNA3.1‑Egr. 1p‑p16 plasmid + GKRS groups than that in the remaining groups 28 days following the GKRS management. The p16mRNA expression was noted in both of the pcDNA3.1‑Egr. 1p‑p16 plasmid group and the pcDNA3.1‑Egr. 1p‑p16 plasmid + GKRS with marginal‑dose of 20 Gy group. The level of messenger ribonucleic acid expression was higher in the pcDNA3.1‑Egr. 1p‑p16 plasmid + GKRS with the marginal‑dose of 20 Gy group, with a markedly increased apoptotic and necrotic cells, than that in the pcDNA3.1‑Egr. 1p‑p16 plasmid group. Conclusions: In animal studies, pcDNA3.1‑Egr. 1p‑p16 in combination with GKRS is a preferable management option for the GBM to the sole use of GKRS or gene therapy. It may be a novel approach for the treatment of human patient with GBM.
Access this article online Quick Response Code:
Website: www.neurologyindia.com PMID: *** DOI: 10.4103/0028-3886.121917
Neurology India | Sep-Oct 2013 | Vol 61 | Issue 5
Key words: Gamma knife, gene therapy, glioblastoma
multiforme, recombinant plasmid pcDNA3.1‑Egr. 1p‑p16
Introduction The annual incidence rate of glioblastoma is about 5/100,000 persons, 60‑70% of which are glioblastoma multiforme (GBM). [1] Integrated treatment of 491
Wenke, et al.: Treatment of glioblastoma multiforme with gamma knife and PcDNA3.1‑Egr. 1p‑p16
surgical resection combined with radiotherapy and chemotherapy is the mainstream therapeutic solution in GBM. However, high postoperative recurrence and poor prognosis, which is likely due to the infiltrative growth of the tumor and it also effects the quality of life the patients. Combined usage of tumor suppressor gene p16 with radiation‑induced promoter Egr‑1p is new approach of gene and radiation therapy for GBM. In our experimental study, pcDNA3.1‑Egr. 1p‑p16 plasmid is innovatively combined with gamma knife radio‑surgery (GKRS) for the treatment of brain GBM, which has not been reported before. Our study is to investigate the possible synergistic effect of the combined treatment of GKRS and gene therapy for GBM and to explore the role of GKRS for the temporal and spatial regulation of the gene expression.
Materials and Methods Reagents and apparatus 1. pcDNA3.1‑Egr. 1p‑p16 plasmid was constructed by GenScript Co. Ltd.; 2. Human GBM cell line T98G was provided by State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University; 3. 4‑6 weeks aged BALB/c‑nu/nu female nude mice with the weight of approximately 20 g were purchased from Chengdu Dashuo Biosciences Co. Ltd.; 4. Plasmid Midi Kit was from Qiagen Co. Ltd.; 5. In situ terminal‑deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) array kit was from Roche Co. Ltd.; 6. Lipofectamine 2000 was from Invitrogen Co. Ltd.; 7. Magnetic resonance imaging scanner, SIEMENS Sonata 1.5T; 8. Gamma Knife, Leksell Gamma Knife Model C, Elekta Co. Ltd. Experiment methods Cell thawing and cultivation Frozen cells were removed from liquid nitrogen and quickly placed in 37°C water bath for rewarming and thawing. The cells were washed once with Dulbecco’s modified eagle medium (DMEM) and then centrifuged at 1500 rpm for 5 min. The supernatant was removed and the cells were then cultivated on the complete DMEM at 37°C in a 5% CO2 incubator. Cell passage The medium was removed and the cells were washed twice with pre‑warmed serum‑free DMEM and digested by 1% trypsin‑ethylene diamine tetraacetic acid. The digestion was terminated by adding complete DMEM when the cells became smaller and round under the microscope. After the cells were blown 492
scattered, the liquid was collected and centrifuged at 1500 rpm for 5 min. The cells were re‑suspended in the complete medium after precipitation and divided into flasks for continuous cultivation. When grew to a certain quantity, the well grown cells were collected and washed with sterile phosphate buffer solution (PBS). The cells were counted on a counting chamber under microscope to adjust the concentration to 5 × 107 cells/ml. Preparation of plasmid The bacterial strain for converting the pcDNA3.1‑Egr. 1p‑p16 recombinant plasmid was inoculated in the Luria‑Bertani medium and then cultivated at 37°C on a shaker overnight. The plasmid was extracted by the alkaline lysis method with modification. Restriction enzyme digestion was performed on collected samples for confirmation. Animal model establishment A total of 70, 4‑6 weeks aged BALB/c‑nu/nu nude mice, weighing approximately 20 g each, were used for the experimental study. Each nude mouse was inoculated with 5 × 106 T98G cells (0.1 ml) subcutaneously under the right axilla. An equal volume of PBS was subcutaneously injected under the right axilla of the control nude mice. The animals were treated i when the tumors in the mice of the experimental group grew up to 5 mm in diameter. The grouping and treatments are detailed in Table 1. Post‑modeling treatments The liposome‑encapsulated pcDNA3.1‑Egr. 1p‑p16 recombinant plasmid was transfected to the tumor cells by intra‑tumor injection. The nude mice were anesthetized by intra‑peritoneal injection of 3% pentobarbital sodium (40 mg/kg) 48 h after transfection. The animal was fixed on a previously prepared polymethyl methacrylate made fixation device. Then the device was fixed onto the Leksell stereotactic head frame [Figure 1]. The radiation procedure was finished using Leksell Gamma Knife Model C under the guidance of magnetic resonance imaging (Siemens sonata 1.5T). According to the grouping rules, the tumor was covered with the established isodose line [Figure 2]. The radiation doses are detailed in Table 1. Observation of anti‑tumor effect Tumor volume was recorded by measuring its length (a) and width (b) using a vernier caliper every 4 days after the GKRS. Tumor growth rate and volume were calculated based on the formulas: tumor growth rate f = post‑radiation tumor volume/pre‑radiation tumor volume; tumor volume V (mm3) = (a × b2)/2. The data were presented in x ± s. To compare the tumor volume changes, a t‑test was used. Statistical significance was considered at a P ≤ 0.05. Neurology India | Sep-Oct 2013 | Vol 61 | Issue 5
Wenke, et al.: Treatment of glioblastoma multiforme with gamma knife and PcDNA3.1‑Egr. 1p‑p16
Figure 2: Gamma knife treatment protocol: the tumor foci was covered by the isodose line
Figure 1: Gamma knife treatment status
Table 1: Grouping of experiment animal and tumor growth rate
Group
Group code
No. of animal
Radiation Null plasmid group+radiation Plasmid group 1
R Palone+R
10 10
P1
10
Plasmid group 2
P2
10
Plasmid group 1+20 Gy radiation Plasmid group 2+20 Gy radiation Control group
P1+R
10
P2+R
10
Control
10
Treatment
Tumor growth rate
Sole 20 Gy marginal‑dose radiation Intra‑tumoral injection of 20 µg pcDNA 3.1+plasmid encapsulated in 50 µl liposome+20 Gy marginal‑dose radiation Intra‑tumoral injection of 20 µg pcDNA3.1‑p16 plasmid encapsulated in 50 µl liposome Intra‑tumoral injection of 20 µg pcDNA3.1‑Egr. 1p‑p16 plasmid encapsulated in 50 µl liposome Intra‑tumoral injection of 20 µg pcDNA3.1‑p16 plasmid encapsulated in 50 µl liposome+20 Gy marginal‑dose radiation Intra‑tumoral injection of 20 µg pcDNA3.1‑Egr. 1p‑p16 plasmid encapsulated in 50 µl liposome+20 Gy marginal‑dose radiation Intra‑tumoral injection of 50 µl PBS, without radiation
3.51±0.88 3.40±0.7 3.83±1.11 3.85±0.65 1.95±0.35 1.00±0.21 5.44±1.02
Each group was individually compared with the control group and the differences in tumor growth rate were statistically significant (P
